Circulating Progenitor Cells and Cardiovascular Disease Arshed A. Quyyumi, MD Professor of Medicine...

Circulating Progenitor Cells and Cardiovascular Disease

Arshed A. Quyyumi, MD Professor of Medicine Emory Clinical Cardiovascular Research Institute (ECCRI)Emory University School of MedicineAtlanta, GA

Grant support: National Institutes of Health, American Heart Association, Woodruff Fund, Emory Heart and Vascular Center,Sanofi Aventis, Novartis, Lilly, Pfizer, Forest,Amgen, GenzymeAdvisory Boards:Amorcyte/Neostem, Genway/FirstmarkSoteriaStemedica


Circulating progenitor cellsCD34+ cell populationsCirculating progenitor cells and genderCirculating progenitor cells and ischemic

syndromesCirculating progenitor cells and CVD

outcomesCD34+ cells as therapy for vascular diseases


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Adult Bone Marrow Stem Cell Plasticity

EndodermalProgenitor Cells

Bone MarrowStem Cells

EctodermalProgenitor Cells

MesodermalProgenitor Cells

Neural cellsEpidermal cells

Hepatocytes

Hematopoeitic cells

Endothelial Progenitor Cells

Myocytes (Skeletal) (Cardiac)

Osteocytes, Chondrocytes

Stromal orMesenchymal MAPC

Blood cells

Resident stem cells:Heart, skeletal muscle,Adipose tissue, brain,Lung etc.

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0 50K 100K 150K 200K 250KFSC-A

0

50K

100K

150K

200K

250K

SS

C-A

31.8

0 102 103 104 105

<FITC-A>: CD34

0

50K

100K

150K

200K

250K

SS

C-A

0.113

0 102 103 104 105

<PE-A>: VEGF-R2

0

102

103

104

105

<A

PC

-A>

: CD

133

35.2 0.585

1.8562.4

Gated on CD34+ within MNCs

Within CD34+ gate, 4 quadrants for marker CD133 and VEGF-2R

Gated on MNCsCD45med

Flow Cytometry

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Fluorescent activated cell sorting (FACS) analysis for bone marrow derived progenitor cell populations

CD34: Hematopoietic stem cell

CD133: Immature progenitorsCD133+/VEGF2R+:

Differentiates between immature and mature endothelial PCs

CD34+/CD133+/VEGF2R+: Presumed ‘EPC’ enriched

CXCR4: Epitope that is associated with homing to areas of ischemia that express SDF-1

Peichev M et al Blood 2000; 95:952Hirschi KK. et al ATVB 2008;28:1584

Cell Type: Isolated CD34⁺Cells Most Able to Improve Perfusion, Prevent Apoptosis and Rescue Hibernating Cardiomyocytes

13Kawamoto et al., Circulation 2006;114;2163-2169

PBS = Phosphate-buffered salineloMNCs = 5x10^5 MNChiMNCs = contains 5x10^5 CD34+ cells within MNCsCD34+ = 5x10^5 CD34+ cells

Capillary Density (perfusion) is greatest in CD34+ cell cohort, and this correlates with decreased incidence of fibrosis. Effect increases with dose.

CD34⁺ Cells Exhibit Increased Potency and Safety for Therapeutic Neovascularization after AMI Compared with Total Mononuclear Cells in Nude Rats:

Endothelial progenitor cell therapy for acute myocardial infarction

Cell types: bone marrow mononuclear cells, CD34+, etc.CD34+ cells constitute 0.1 to 0.2% of bone marrow mononuclear

cells

• FDG labeled bone marrow mononuclear cells and injected intracoronary in post MI patients

• Uptake of BM mononuclear cells: 1.3 to 2.6% in MI region

• Uptake of CD34+ cells: 14-39% in MI region

BM mononuclear cells

CD34+ cells

Hofmann et al Circ 2005;111:2198-2202

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Men: r=-0.16, p=0.011Women: r=-0.13, p=0.003

Men: r=-0.19, p=0.003 Women: r=-0.14, p=0.001

Men: r=-0.1, p=0.05Women: r=-0.04, p=0.4

Men: r=-0.1, p=0.09Women: r=-0.05, p=0.2

Figure3: Age-related changes in circulating PC populations in men (green) and women (blue) Hematopoietic (top) and endothelial-enriched PCs (bottom)

Circulating progenitor cellsCD34+ cell populationsCirculating progenitor cells and ischemic




90 ACS patients (mean age 65±15 yrs, 73% male, 10% STEMI, 76% NSTEMI, and 13% unstable angina) Blood samples were obtained at the time of cardiac catheterization for enumeration of CPCs as CD45dim cells using flow cytometry.

An age- and gender-matched (1:2) cohort of stable CAD patients were randomly selected as a control group

Circulating progenitor cells in acute coronary syndromes: comparison with stable CAD

*

*

Comparison of VEGF2R-expressing CPCs between stable CAD and ACS categories

*

*

Mental Stress Standardized public

speaking task Role playing a difficult

interpersonal situation where a close relative in a nursing home is being mistreated.

Hemodynamic and electrocardiographic monitoring

Mental stress Physical stress

MentalStress

Rest

Physical Stress

Rest

Results Effect of mental stress on acute mobilization of progenitor cells

• MS challenge provoked an average 21% increase in the number of circulating CD34+/VEGF2R+/CXCR4+ cells

Relationship between progenitor cells and ischemia during mental stress

Patients with mental stress ischemia had higher circulating number of CXCR4-expressing cells at baseline

Patients:• 160 women enrolled in the

WISE-CVD Study with ischemia during stress testing

• No obstructive CADProtocol:• CFR measured as ratio of

hyperemic average peak velocity (APV) in response to intracoronary adenosine to baseline APV

• Lower CFR with adenosine correlated significantly with higher levels of CD34+, CD34+/CD133+ and CD34+/CXCR4+ cells

Circulating Progenitor Cells and Coronary Microvascular Dysfunction: Results from the NHLBI-Sponsored Women’s Ischemia Syndrome Evaluation - Coronary

Vascular Dysfunction (WISE-CVD) Study

Patients:• 160 women enrolled in the

WISE-CVD Study with ischemia during stress testing

• No obstructive CADProtocol:• Microvascular endothelial

function measured as the CBF response to intracoronary acetylcholine (n=48)

• Lower CBF with acetylcholine correlated significantly with higher CD34+/CXCR4+ and CD34+/VEGF+.

Circulating Progenitor Cells and Coronary Microvascular Dysfunction: Results from the NHLBI-Sponsored Women’s Ischemia Syndrome Evaluation - Coronary

Vascular Dysfunction (WISE-CVD) Study

• ACS is associated with mobilization of VEGF2R and CXCR4 expressing PCs

• Subjects with microvascular coronary ischemia with reduced flow reserve and endothelial dysfunction have higher circulating PC subsets

• PC expressing CXCR4, denoting cells with a capacity to home to areas of ischemia, are increased in those who develop ischemia during MS.

• This suggests that even mild ischemia during daily living may stimulate PC mobilization.

Progenitor Cells and ischemic syndromes

Circulating progenitor cellsCD34+ cell populationsCirculating progenitor cells and sub-clinical

vascular diseaseCirculating progenitor cells and ischemic




Risk factors, Vascular Injury, and Regenerative Capacity

Risk Factors

Endothelial injury

Endothelial dysfunction, Arterial stiffness

AtherogenesisVascular Repair

Progenitor Cells

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Relationship Between circulating Progenitor Cell Counts and Long term CVD outcomes (Death/MI)

• Patients undergoing coronary angiography: Treated with guideline based therapies

• 502 patients in a Discovery cohort ;• 403 patients in a Validation cohort.• Total Pooled 905; age 63yrs; 65% male

• PCs were enumerated by flow cytometry as CD45med+ blood mononuclear cells expressing CD34, CD133, VEGFR2 and/or CXCR4

• Followed patients in each cohort for a mean of 2.7 & 1.2 years, for the primary endpoint of death or myocardial infarction (MI).

92 death/MI (10%)

Higher counts of CD34+ and CD34+/CD133+ cells correlated with: younger age (p<0.001 both),

male gender (p=0.04 and p<0.011) higher GFR (p<0.001 both)Higher CD34+/CD133+ also with greater BMI (p<0.001).

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Relationship Between circulating Progenitor Cell Counts and Event Free Survival (Major Events - Death/MI)

C statistic improved from 0.713 to 0.752, p=0.024 for CD34/133+

CD

34

+

39


• Patients undergoing coronary angiography

• 502 patients in a Discovery cohort ;

• 403 patients in a Validation cohort.

• Total Pooled 905; age 63yrs; 65% male

• PCs were enumerated by flow cytometry as CD45med+ blood mononuclear cells expressing CD34, CD133, VEGFR2 and/or CXCR4

• Followed patients in each cohort for a mean of 2.7 & 1.2 years, for the primary endpoint of death or myocardial infarction (MI).

92 death/MI

Cell Population Outcome Low v High ROCCD34+ Death/MI

DeathCV Death

2.76 (1.65-4.61)2.60 (1.45-4.66)2.35 (1.28-4.35)

CD34+/133+ Death/MIDeath

CV Death

2.98 (1.68-5.30)2.66 (1.40-5.03)2.49 (1.28-4.86)

CD34+/VEGF+ Death/MIDeath

CV Death

1.01(0.60-1.69)1.18 (0.65-2.14)1.34 (0.71-2.50)

CD34+/133+/VEGF Death/MIDeath

CV Death

0.97 (0.59-1.59)0.83 (0.47-1.47)0.83(0.45-1.50)

CD34+/CXCR4+ Death/MIDeath

CV death

2.50 (1.19-5.23)2.15 (1.00-4.62)1.82(0.82-3.96)

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Relationship Between circulating Progenitor Cell Counts and Event Free Survival (Major Events - Death/MI)

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Conclusion: Low levels of circulating PCs, defined as co-expression of CD34 and CD133 and CXCR4 epitopes are robustly associated with risk of future death/MI in patients with CAD.

Implications; (1) CD34+/CD133+ cells that are enriched for bone marrow-derived

hematopoietic and endothelial progenitors, may represent an index of global regenerative potential

(2) PCs protect or regenerate damaged endothelium by local or systemic paracrine effects,

(3) The predictive value of PCs as risk markers was equal or greater than conventional risk factors such as smoking.

RepairPCs

AtherosclerosisIncidence

Age or Risk Factor Years

RiskRepair

Risk

PCs

Arshed Quyyumi Riyaz Patel Danny Eapen Nima Ghassemzadeh Ronnie Ramadan Ibhar Al Mheid Girum Mekonnen Joseph Poole Robert Neuman Pankaj Manocha Hatem Kassem Alanna Morris Ayaz Rahman Saurabh Dhawan Salman Sher Ying Liu Nino Kavtaradze Elizabeth Rocco Sherri Mcdonald

Cath Lab Attendings and Staff Fellows/ Research Volunteers

AcknowledgementsEmory Clinical Cardiovascular Research institute

Viola Vaccarino Emir Veledar A Maziar Zafari Laurence Sperling Edmund Waller Qunna Li

DeCode Genetics Dean Jones PhD (Metabolomics) Charles Searles (miRNA) Greg Gibson PhD (Transcriptomics)

Circulating Progenitor Cells and Cardiovascular Disease Arshed A. Quyyumi, MD Professor of Medicine...

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